In vitro induction of tetraploidy and its effects on phenotypic variations in Populus hopeiensis.

BACKGROUND: Artificial induction of polyploidy is the most common and effective way to improve the biological properties of Populus and develop new varieties of this tree. In this study, in order to confirm and expand earlier findings, we established a protocol using colchicine and based on an efficient shoot regeneration system of leaf blades to induce tetraploidy in vitro in three genotypes from diploid Populus hopeiensis. The stomatal characteristics, leaf blade size, and growth were evaluated for diploids and tetraploids of three genotypes. RESULTS: We found that genotype, preculture duration, colchicine concentration, and colchicine exposure time had highly significant effects on the tetraploid induction rate. The optimal protocol for inducing tetraploidy in P. hopeiensis was to preculture leaf blades for 7 days and then treat them for 4 days with 40 mg/L colchicine. The tetraploid induction rates of genotypes BT1, BT3, and BT8 were 21.2, 11.4 and 16.7%, respectively. A total of 136 tetraploids were identified by flow cytometry analysis and somatic chromosome counting. The stomatal length, width, and density of leaf blades significantly differed between diploid and tetraploid plants. Compared with their diploid counterparts, the tetraploids produced larger leaf blades and had a slower growth rate. Our findings further document the modified morphological characteristics of P. hopeiensis following whole-genome duplication (e.g., induced tetraploidy). CONCLUSIONS: We established a protocol for in vitro induction of tetraploidy from diploid leaf blades treated with colchicine, which can be applied to different genotypes of P. hopeiensis.

Regional testing of triploid hybrid clones of populus tomentosa.

BACKGROUND: Triploid Populus tomentosa, a timber tree species, has been widely planted in northern China owing to its potential high yields and high wood quality. Though genetic variances in growth traits and wood properties have been reported across several planting sites, regional testing of triploid hybrid clones of P. tomentosa has not been conducted on a large scale. RESULTS: Ten 5-year clonal trials were used to evaluate the inheritance of growth traits, to determine suitable deployment zones, and to identify optimal triploid clones at each experimental site to determine the clones that would be suitable at all sites. A total of 2,430 trees from nine triploid hybrid clones were sampled during the ten trials. The clonal and site effects and clone × site interactions were highly significant (P < 0.001) for all the studied growth and yield traits. The estimated repeatability of means for diameter at breast height (DBH) and tree height (H) was 0.83, which was slightly higher than for stem volume (SV) and estimated stand volume (ESV) (0.78). The Weixian (WX), Gaotang (GT), and Yanzhou (YZ) sites were each considered to be suitable deployment zones, and the Zhengzhou (ZZ), Taiyuan (TY), Pinggu (PG), and Xiangfen (XF) sites were found to be the optimal deployment zones. The TY and ZZ sites were the best discriminative environments, and the GT and XF sites were the best representative environments. GGE pilot analysis revealed that yield performance and stability were significantly different among all the studied triploid hybrid clones across the ten test sites. It was therefore necessary to develop a suitable triploid hybrid clone that could do well at each site. Taking into account both yield performance and stability, the triploid hybrid clone S2 was determined to be an ideal genotype. CONCLUSIONS: For triploid hybrid clones, the WX, GT, and YZ sites represented suitable deployment zones and the ZZ, TY, PG, and XF sites represented optimal deployment zones. Yield performance and stability were significantly different among all the studied triploid hybrid clones across the ten test sites. Developing a suitable triploid hybrid clone that could do well at all sites was therefore desirable.

Microsporogenesis in the triploid hybrid 'Beilinxiongzhu 1#' and detection of primary trisomy in 2x × 3 × Populus hybrids.

BACKGROUND: Primary trisomy is a powerful genetic tool in plants. However, trisomy has not been detected in Populus as a model system for tree and woody perennial plant biology. RESULTS: In the present study, a backcross between Populus alba × Populus glandulosa 'YXY 7#' (2n = 2x = 38) and the triploid hybrid 'Beilinxiongzhu 1#' (2n = 3x = 57) based on the observation of microsporogenesis and an evaluation of the variations in pollen was conducted to create primary trisomy. Many abnormalities, such as premature migration of chromosomes, lagging of chromosomes, chromosome bridges, asymmetric separation, micronuclei, and premature cytokinesis, have been detected during meiosis of the triploid hybrid clone 'Beilinxiongzhu 1#'. However, these abnormal behaviors did not result in completely aborted pollen. The pollen diameter of the triploid hybrid clone 'Beilinxiongzhu 1#' is bimodally distributed, which was similar to the chromosomal number of the backcross progeny. A total of 393 progeny were generated. We provide a protocol for determining the number of chromosomes in aneuploid progeny, and 19 distinct simple sequence repeat (SSR) primer pairs covering the entire Populus genome were developed. Primary trisomy 11 and trisomy 17 were detected in the 2x × 3 x hybrid using the SSR molecular markers and counting of somatic chromosomes. CONCLUSIONS: Nineteen distinct SSR primer pairs for determining chromosomal number in aneuploid individuals were developed, and two Populus trisomies were detected from 2x × 3 x hybrids by SSR markers and somatic chromosome counting. Our findings provide a powerful genetic tool to reveal the function of genes in Populus.

SCL14 Inhibits the Functions of the NAC043-MYB61 Signaling Cascade to Reduce the Lignin Content in Autotetraploid Populus hopeiensis.

Whole-genome duplication often results in a reduction in the lignin content in autopolyploid plants compared with their diploid counterparts. However, the regulatory mechanism underlying variation in the lignin content in autopolyploid plants remains unclear. Here, we characterize the molecular regulatory mechanism underlying variation in the lignin content after the doubling of homologous chromosomes in Populus hopeiensis. The results showed that the lignin content of autotetraploid stems was significantly lower than that of its isogenic diploid progenitor throughout development. Thirty-six differentially expressed genes involved in lignin biosynthesis were identified and characterized by RNA sequencing analysis. The expression of lignin monomer synthase genes, such as PAL, COMT, HCT, and POD, was significantly down-regulated in tetraploids compared with diploids. Moreover, 32 transcription factors, including MYB61, NAC043, and SCL14, were found to be involved in the regulatory network of lignin biosynthesis through weighted gene co-expression network analysis. We inferred that SCL14, a key repressor encoding the DELLA protein GAI in the gibberellin (GA) signaling pathway, might inhibit the NAC043-MYB61 signaling functions cascade in lignin biosynthesis, which results in a reduction in the lignin content. Our findings reveal a conserved mechanism in which GA regulates lignin synthesis after whole-genome duplication; these results have implications for manipulating lignin production.

Heat shock-induced failure of meiosis I to meiosis II transition leads to 2n pollen formation in a woody plant.

The formation of diploid gametes through chromosome doubling is a major mechanism of polyploidization, diversification, and speciation in plants. Unfavorable climate conditions can induce or stimulate the production of diploid gametes during meiosis. Here, we demonstrated that heat shock stress (38°C for 3 or 6 h) induced 2n pollen formation, and we generated 42 triploids derived from heat shock-induced 2n pollen of Populus canescens. Meiotic analysis of treated pollen mother cells revealed that induced 2n pollen originated from the complete loss of meiosis II (MII). Among the 42 triploids, 38 triploids derived from second division restitution (SDR)-type 2n pollen and 4 triploids derived from first division restitution-type 2n pollen were verified using simple sequence repeats (SSR) molecular markers. Twenty-two differentially expressed genes related to the cell cycle were identified and characterized by expression profile analysis. Among them was POPTR_0002s08020g (PtCYCA1;2), which encodes a type A Cyclin CYCA1;2 that is required for the meiosis I (MI) to MII transition. After male flower buds were exposed to heat shock, a significant reduction was detected in PtCYCA1;2 expression. We inferred that the failure of MI-to-MII transitions might be associated with downregulated expression of PtCYCA1;2, leading to the formation of SDR-type 2n pollen. Our findings provide insights into mechanisms of heat shock-induced 2n pollen formation in a woody plant and verify that sensitivity to environmental stress has evolutionary importance in terms of polyploidization.

In vitro octaploid induction of Populus hopeiensis with colchicine.

BACKGROUND: Autopolyploids, especially artificial lines, provide model systems for understanding the mechanisms of gene dosage effects on trait variation owing to their relatively uniform genetic background. Here, a protocol for in vitro octaploid induction of Populus hopeiensis from leaf blades with colchicine treatment was established through investigation of the effects of different pre-culture durations, colchicine concentrations, and exposure times. RESULTS: We found that pre-culture duration, colchicine concentration, and exposure time had significant effects on the survival rate, shoot regeneration rate, and octaploid induction rate of P. hopeiensis leaf blades. The highest octaploid induction rate (8.61%) was observed when leaf blades pre-cultured for 9 days were treated for 4 days with 100 µM colchicine. The ploidy level of all regenerated plantlets was analyzed by flow cytometry and further confirmed by chromosome counting. A total of 14 octaploids were obtained. The stomatal length, width, and density of leaf blades significantly differed between tetraploid and octaploid plants. Compared with diploid and tetraploid plants, octaploids had a slower growth rate, smaller leaf blade size, and shorter internodes. CONCLUSIONS: We established an effective protocol for inducing octaploids in vitro from autotetraploid P. hopeiensis leaf blades by colchicine treatment.

Populus root exudates are associated with rhizosphere microbial communities and symbiotic patterns.

Introduction: Microbial communities in the plant rhizosphere are critical for nutrient cycling and ecosystem stability. However, how root exudates and soil physicochemical characteristics affect microbial community composition in Populus rhizosphere is not well understood. Methods: This study measured soil physiochemistry properties and root exudates in a representative forest consists of four Populus species. The composition of rhizosphere bacterial and fungal communities was determined by metabolomics and high-throughput sequencing. Results: Luvangetin, salicylic acid, gentisic acid, oleuropein, strigol, chrysin, and linoleic acid were the differential root exudates extracted in the rhizosphere of four Populus species, which explained 48.40, 82.80, 48.73, and 59.64% of the variance for the dominant and key bacterial or fungal communities, respectively. Data showed that differential root exudates were the main drivers of the changes in the rhizosphere microbial communities. Nitrosospira, Microvirga, Trichoderma, Cortinarius, and Beauveria were the keystone taxa in the rhizosphere microbial communities, and are thus important for maintaining a stable Populus microbial rhizosphere. The differential root exudates had strong impact on key bacteria than dominant bacteria, key fungi, and dominant fungi. Moreover, strigol had positively effects with bacteria, whereas phenolic compounds and chrysin were negatively correlated with rhizosphere microorganisms. The assembly process of the community structure (keystone taxa and bacterial dominant taxa) was mostly determined by stochastic processes. Discussion: This study showed the association of rhizosphere microorganisms (dominant and keystone taxa) with differential root exudates in the rhizosphere of Populus plants, and revealed the assembly process of the dominant and keystone taxa. It provides a theoretical basis for the identification and utilization of beneficial microorganisms in Populus rhizosphere.

Gibberellins as a novel mutagen for inducing 2n gametes in plants.

The plant hormone gibberellin (GA) regulates many physiological processes, such as cell differentiation, cell elongation, seed germination, and the response to abiotic stress. Here, we found that injecting male flower buds with exogenous gibberellic acid (GA3) caused defects in meiotic cytokinesis by interfering with radial microtubule array formation resulting in meiotic restitution and 2n pollen production in Populus. A protocol for inducing 2n pollen in Populus with GA3 was established by investigating the effects of the dominant meiotic stage, GA3 concentration, and injection time. The dominant meiotic stage (F = 41.882, P < 0.001) and GA3 injection time (F = 172.466, P < 0.001) had significant effects on the frequency of induced 2n pollen. However, the GA3 concentration (F = 1.391, P = 0.253) did not have a significant effect on the frequency of induced 2n pollen. The highest frequency of GA3-induced 2n pollen (21.37%) was observed when the dominant meiotic stage of the pollen mother cells was prophase II and seven injections of 10 µM GA3 were given. Eighteen triploids were generated from GA3-induced 2n pollen. Thus, GA3 can be exploited as a novel mutagen to induce flowering plants to generate diploid male gametes. Our findings provide some new insight into the function of GAs in plants.

Genotype-environment interaction and stability of fiber properties and growth traits in triploid hybrid clones of Populus tomentosa.

BACKGROUND: Clones provide a sensitive method for evaluating genotypic stability and detecting genotype-environment (G × E) interactions because of non-additive genetic effects among clones and there being no genetic effect among ramets of an ortet. With this study, we aimed to confirm and expand earlier findings, estimate stability parameters, and provide accurate estimates of clonal repeatabilities and genetic gains for a triploid breeding program of P. tomentosa Carr. RESULTS: Six 5-year-old clonal trials established in Northern China were used to determine the clonal variation, clone × site interactions, and the stability parameters of fiber properties of wood and growth traits. 360 trees from ten hybrid clones were collected from six sites. The clonal and site effects had a highly significant effect (P < 0.001) for all studied traits. While the clone × site interactions had a highly significant effect (P < 0.001) on fiber length (FL), coarseness (C), and tree growth (tree height [H], diameter at breast height [DBH] and stem volume [SV]), and a moderate effect (P < 0.05) on fiber width (FW) and fiber length/width (FL/W). For FL and SV, most of the triploid hybrid clones had higher reaction norms to the improvement in growth conditions and higher phenotypic plasticity. The estimated clonal repeatability of FW (0.93) was slightly higher than for FL (0.89), FL/W (0.83), C (0.91), DBH (0.76), H (0.85), and SV (0.80). Three clonal testing sites were sufficient to estimate quantitative parameters of fiber properties, however, more clonal testing sites would help improve the accuracy of quantitative parameters of the growth traits. CONCLUSIONS: Our results highlight that accurate estimation of quantitative parameters for growth traits in triploid hybrid clones of P. tomentosa requires more clonal testing sites than the fiber properties.

Effects of Colchicine on Populus canescens Ectexine Structure and 2n Pollen Production.

Triploid breeding is a central way to improve growth traits, timber quality, and stress resistance in Populus. In the present study, the morphology and viability of colchicine-induced 2n pollen, triploid production by crossing induced 2n pollen, and identification of genetic constitution of colchicine-induced 2n pollen were conducted in Populus canescens based on optimizing technology for inducing chromosome doubling in pollen. We found that the meiotic stage, injection time, and the interaction between the meiotic stage and injection time had highly significant effects on the 2n pollen production rate. The most effective treatment for inducing 2n pollen was to give 11 injections of 0.5% colchicine solution when pollen mother cells (PMCs) were at the pachytene stage. The highest 2n pollen production rate was 30.27 ± 8.69%. Colchicine occasionally affected ectexine deposition, and some narrow furrows were detected in the ectexine structure. However, no significant difference was observed in the pollen germination rate between natural 2n pollen and colchicine-induced 2n pollen. Moreover, 5 triploids derived from FDR-type 2n pollen were generated by crossing induced 2n pollen, suggesting that colchicine does not eliminate the function of colchicine-induced 2n pollen. However, slower growth of 2n pollen tubes was responsible for a lower triploid production rate.

An extraordinarily stable karyotype of the woody Populus species revealed by chromosome painting.

The karyotype represents the basic genetic make-up of a eukaryotic species. Comparative cytogenetic analysis of related species based on individually identified chromosomes has been conducted in only a few plant groups and not yet in woody plants. We have developed a complete set of 19 chromosome painting probes based on the reference genome of the model woody plant Populus trichocarpa. Using sequential fluorescence in situ hybridization we were able to identify all poplar chromosomes in the same metaphase cells, which led to the development of poplar karyotypes based on individually identified chromosomes. We demonstrate that five Populus species, belonging to five different sections within Populus, have maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements were observed on any of the 19 chromosomes among the five species. Thus, the chromosomal synteny in Populus has been remarkably maintained after nearly 14 million years of divergence. We propose that the karyotypes of woody species are more stable than those of herbaceous plants since it may take a longer period of time for woody plants to fix chromosome number or structural variants in natural populations.

Amplification and adaptation of centromeric repeats in polyploid switchgrass species.

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats from a single satellite repeat family. Why centromeres are dominated by a single satellite repeat and how the satellite repeats originate and evolve are among the most intriguing and long-standing questions in centromere biology. We identified eight satellite repeats in the centromeres of tetraploid switchgrass (Panicum virgatum). Seven repeats showed characteristics associated with classical centromeric repeats with monomeric lengths ranging from 166 to 187 bp. Interestingly, these repeats share an 80-bp DNA motif. We demonstrate that this 80-bp motif may dictate translational and rotational phasing of the centromeric repeats with the cenH3 nucleosomes. The sequence of the last centromeric repeat, Pv156, is identical to the 5S ribosomal RNA genes. We demonstrate that a 5S ribosomal RNA gene array was recruited to be the functional centromere for one of the switchgrass chromosomes. Our findings reveal that certain types of satellite repeats, which are associated with unique sequence features and are composed of monomers in mono-nucleosomal length, are favorable for centromeres. Centromeric repeats may undergo dynamic amplification and adaptation before the centromeres in the same species become dominated by the best adapted satellite repeat.

High temperature exposure did not affect induced 2n pollen viability in Populus.

High temperature exposure is widely used as a physical mutagenic agent to induce 2n gametes in Populus. However, whether high temperature exposure affects induced 2n pollen viability remains unknown. To clarify whether high temperature exposure affected the induced 2n pollen viability, 2n pollen induced by 38 and 41 °C temperatures, pollen morphology, 2n pollen germination in vitro, and crossing induced 2n pollen with normal gametes to produce a triploid was, based on observations of meiosis, conducted in Populus canescens. We found that the dominant meiotic stages (F = 56.6, p < .001) and the treatment duration (F = 21.4, p < .001) significantly affected the occurrence rate of induced 2n pollen. A significant decrease in pollen production and an increase in aborted pollen were observed (p < .001). High temperature sometimes affected in ectexine deposition and some narrow furrows were also analysed via details of ectexine structure. However, no significant difference in 2n pollen germination rate was observed between natural 2n pollen (26.7%) and high-temperature-induced 2n pollen (26.2%), and 42 triploids were created by crossing high-temperature-induced 2n pollen, suggesting that 38 and 41 °C temperatures exposure will not result in dysfunctional induced 2n pollen.

Embryo sac chromosome doubling in Populus alba × P. glandulosa induced by high temperature exposure to produce triploids.

To determine the effects of the hours after pollination and the treatment durations on triploid production and reveal the effective stages of embryo sac chromosome doubling by high temperature exposure. At least three catkins were sampled, and 80 ovules were used for the determination of the embryo sac developmental process. Catkins (2-74 h after pollination) were treated to induce embryo sac chromosome doubling. Cytological observations revealed that the embryo sac development was a consecutive and asynchronous process. Fertilization occurred 50 h after pollination. In the offspring seedlings, 167 triploids were detected and the highest efficiency of triploid production was 87.0%. Among all the induced triploids, the most effective treatment period of inducing embryo sac chromosome doubling is from 26 to 50 h after pollination, and 121 triploids were obtained, representing 72.46% of the sum of all triploids. GLM-Univariate analysis indicated significant differences among the hours after pollination (F = 4.516, p = 0.045). However, the differences between the treatment durations (F = 0.077, p = 0.791) were not significant. Correlation analysis between the proportion of each embryo sac's developmental stage and the percentage of triploid production indicated that the third mitotic division may be the most effective stage for 2n female gamete induction.

Genomic abundance is not predictive of tandem repeat localization in grass genomes.

Highly repetitive regions have historically posed a challenge when investigating sequence variation and content. High-throughput sequencing has enabled researchers to use whole-genome shotgun sequencing to estimate the abundance of repetitive sequence, and these methodologies have been recently applied to centromeres. Previous research has investigated variation in centromere repeats across eukaryotes, positing that the highest abundance tandem repeat in a genome is often the centromeric repeat. To test this assumption, we used shotgun sequencing and a bioinformatic pipeline to identify common tandem repeats across a number of grass species. We find that de novo assembly and subsequent abundance ranking of repeats can successfully identify tandem repeats with homology to known tandem repeats. Fluorescent in-situ hybridization shows that de novo assembly and ranking of repeats from non-model taxa identifies chromosome domains rich in tandem repeats both near pericentromeres and elsewhere in the genome.

Transcriptomic changes following synthesis of a Populus full-sib diploid and allotriploid population with different heterozygosities driven by three types of 2n female gamete.

Diploid gametes are usually applied to produce triploids of Populus [originating from first-division restitution (FDR), second-division restitution (SDR), and postmeiotic restitution (PMR) 2n eggs]. Three types of 2n gametes transmitted different parental heterozygosities in Populus. Failed spindle formation and no chromosomal separation to opposite poles during meiosis I mean that FDR 2n gametes carry nonsister chromatids that are potentially heterozygous. By contrast, SDR 2n gametes result from failed sister chromatid separation in meiosis II, and therefore, they carry sister chromatid that are potentially homozygous. Completely homozygous 2n gametes can arise from the PMR mechanism. The alteration of gene expression resulting from allopolyploidization is a prominent feature in plants. We compared gene expression in the full-sib progeny of three allotriploid Populus populations (triploid-F, triploid-S, and triploid-P) with that in its parent species, and their full-sib diploid F1 hybrid. Genome-wide expression level dominance was biased toward the maternal in the diploid F1 hybrid and three allotriploid populations, whereas our data indicated important, but different, effects of the transmission of different heterozygosity by 2n female gametes in the expression patterns of allopolyploids. Because of the higher level of heterozygosity, the triploids had higher rates of non-additive and transgressive expression patterns in the triploid-F than in triploid-S and triploid-P. Compared with diploid F1, about 30-fold more genes (251) were differently expressed in the triploid-F than in the triploid-S (9) and triploid-P (8), respectively. These findings indicate that hybridization and polyploidization have immediate and distinct effects on the large-scale patterns of gene expression, and different effects on the transmission of heterozygosity by three 2n female gametes.

Differential transcriptome analysis between Populus and its synthesized allotriploids driven by second-division restitution.

In this report, we compared transcriptomic differences between a synthetic Populus section Tacamahaca triploid driven by second-division restitution and its parents using a high-throughput RNA-seq method. A total of 4,080 genes were differentially expressed between the high-growth vigor allotriploids (SDR-H) and their parents, and 719 genes were non-additively expressed in SDR-H. Differences in gene expression between the allotriploid and male parent were more significant than those between the allotriploid and female parent, which may be caused by maternal effects. We observed 3,559 differentially expressed genes (DEGs) between the SDR-H and male parent. Notably, the genes were mainly involved in metabolic process, cell proliferation, DNA methylation, cell division, and meristem and developmental growth. Among the 1,056 DEGs between SDR-H and female parent, many genes were associated with metabolic process and carbon utilization. In addition, 1,789 DEGs between high- and low-growth vigor allotriploid were mainly associated with metabolic process, auxin poplar transport, and regulation of meristem growth. Our results indicated that the higher poplar ploidy level can generate extensive transcriptomic diversity compared with its parents. Overall, these results increased our understanding of the driving force for phenotypic variation and adaptation in allopolyploids driven by second-division restitution.

A strategy for characterization of persistent heteroduplex DNA in higher plants.

Heteroduplex DNA (hDNA) generated during homologous recombination (HR) is an important component that shapes genetic diversity in sexually reproducing organisms. However, studies of this process in higher plants are limited. This is because hDNAs are difficult to capture in higher plants as their reproductive developmental model only produces normal gametes and does not preserve the mitotic products of the post-meiotic segregation (PMS) process which is crucial for studying hDNAs. In this study, using the model system for tree and woody perennial plant biology (Populus), we propose a strategy for characterizing hDNAs in higher plants. We captured hDNAs by constructing triploid hybrids originating from a cross between unreduced 2n eggs (containing hDNA information as a result of inhibition chromosome segregation at the PMS stage) with normal male gametes. These triploid hybrids allowed us to detect the frequency and location of persistent hDNAs resulting from HR at the molecular level. We found that the frequency of persistent hDNAs, which ranged from 5.3 to 76.6%, was related to locations of the simple sequence repeat markers at the chromosomes, such as the locus-centromere distance, the surrounding DNA sequence and epigenetic information, and the richness of protein-coding transcripts at these loci. In summary, this study provides a method for characterizing persistent hDNAs in higher plants. When high-throughput sequencing techniques can be incorporated, genome-wide persistent hDNA assays for higher plants can be easily carried out using the strategy presented in this study.

Induction of 2n female gametes in Populus adenopoda Maxim by high temperature exposure during female gametophyte development.

In order to produce triploid plants, 2n female gametes were induced by treating female buds and developing embryo sacs of Populus adenopoda Maxim with high temperature exposure. During megasporogenesis, tests were conducted on the relationship between female gametophyte development and morphological changes of female catkins. In the resulting progeny, 12 triploids were produced, and the highest rate of triploid production was 40%. Cytological observation revealed that the pachytene to diakinesis phase of meiotic stages may be a suitable period for inducing megaspore chromosome doubling through high temperature exposure. On the other hand, catkins of 6-72 h after pollination were treated for inducing embryo sac chromosome doubling. In the offspring seedlings, 51 triploids were detected and the highest efficiency of triploid production was 83.33%. Correlation analysis between the proportion of each embryo sac's developmental stage and the percentage of triploid production indicated that the second mitotic division may be the most effective stage for 2n female gamete induction. Our findings showed that high temperature exposure is an ideal method for 2n female gamete induction. Heterozygous offspring are valuable for breeding programs of P. adenopoda.